肌酸激酶同工酶MB(CK-MB)单克隆抗体制备及检测方法的建立

Preparation of Creatine Kinase Isoenzyme MB(CK-MB)Monoclonal Antibody and Establishment of Detection Method

该文通过制备出特异性高的单克隆抗体,初步建立了双抗夹心ELISA定量测定CK-MB的方法,为CK-MB试剂和原料的国产化奠定重要的基础。以购入的CK-MB抗原为免疫原,对随机选取的5只6~8周龄Balb/c健康雌性小鼠进行免疫。采用有限稀释法、间接ELISA、捕获ELISA方法,最终筛选出了3株能够稳定分泌抗体的细胞株,分别命名为2F6、2H3和2H9,其分泌的单克隆抗体亚型均为IgG3,腹水效价分别为1:102000、1:51200和1:102000。采用亲和层析法对3株杂交瘤细胞产生的小鼠腹水进行纯化,分光光度计测定纯化后的单克隆抗体2H3、2F6、2H9的浓度分别为5 mg/mL、6 mg/mL、6 mg/mL,SDS-PAGE结果表明,成功纯化了小鼠腹水,能够清晰地观察到轻链和重链两条带。Western blot结果表明,单克隆抗体2F6、2H3和2H9均能特异性识别CK-MB蛋白。间接ELISA及捕获ELISA方法检测显示,单克隆抗体2H3只与CK-MB及CK-BB发生捕获ELISA方法反应;单克隆抗体2H9只与CK-MB及CK-BB发生捕获ELISA方法反应;单克隆抗体2F6只与CK-MB及CK-MM发生捕获ELISA方法反应。稳定性实验表明,3株杂交瘤细胞都能稳定分泌抗CK-MB单克隆抗体。高质量单克隆抗体的获得,为建立CK-MB检测方法奠定了基础。

In this paper by preparing monoclonal antibodies with high specificity,a double antibody sandwich ELISA method for quantitative determination of CK-MB was preliminarily established in the present paper,which laid an important foundation for the domestication of CK-MB reagents and raw materials.Five randomly selected Balb/c female mice(6~8 weeks old)were immunized by using the purchased CK-MB as immunogen.By limited dilution method,indirect and capture ELISA method,three cell lines which could secrete antibodies stably were screened,named as 2F6,2H3 and 2H9,respectively.The monoclonal antibody subtypes secreted by these three cell lines were all IgG3,with ascites titers of 1:102000,1:51200and 1:102000,respectively.The ascites of mice produced by three hybridoma cells was purified by affinity chromatography.The concentrations of monoclonal antibodies 2H3,2F6 and 2H9 determined by spectrophotometer were 5 mg/mL,6 mg/mL and 6 mg/mL,respectively.The results of SDS-PAGE showed that the ascites of monoclonal antibody was purified successfully and two bands of light chain and heavy chain could be clearly observed.Results of Western blot showed that monoclonal antibodies 2F6,2H3 and 2H9 could specifically recognize CK-MB protein.Indirect and capture ELISA detection showed that monoclonal antibody 2H3 reacted only with CK-MB and CK-BB capture ELISA;monoclonal antibody 2H9 reacted only with CK-MB and CK-BB capture ELISA;and monoclonal antibody 2F6 reacted only with CK-MB and CK-MM capture ELISA.The stability test showed that all the three hybridoma cells could secrete anti-CK-MB monoclonal antibodies stably.The acquisition of monoclonal antibodies in high quality lays a foundation for the establishment of CK-MB detection method.